Many different types of stress stimulate cellular signalling pathways that result in stabilisation and activation of the tumour suppressor p53 (reviewed in Pluquet and Hainaut, 2001). Stabilisation of p53 is invariably accompanied by extensive post-translational modifications, including phosphorylation and acetylation (see Appella and Anderson, 2001). Mapping the precise relationships between stress stimuli, specific modifications and the stabilisation and activation of p53 has proven extremely difficult; however general patterns and more specific correlations are now established, particularly with regard to phosphorylation of the amino-terminus and acetylation of the carboxy-terminus.
The transcriptional activity of p53, with particular regard to its pro-apoptotic functions, is tightly regulated. Therefore if acetylation functions to activate p53 transcriptional activity, a logical assumption would be that p53 acetylation is subject to negative control.
Until recently, deacetylation of p53 was only known to be performed by members of the trichostatin A-sensitive histone deacetylase (HDAC) class I family (Juan et al., 2000; Luo et al., 2000). Indeed, mounting evidence suggests that p53 utilises these HDACs to repress specific promoters (Murphy et al., 1999). More recently, the human sirtuin SIRT1 (Frye, 1999) has been identified as a bona fide p53 deacetylase (Luo et al., 2001; Vaziri et al., 2001).
The sirtuins are a ubiquitous gene family found throughout eukarya and prokarya, defined by conserved ˜250 amino acid core domain. Many of the sirtuins are NAD-dependent deacetylases [‘NDAC’ ].
The function of Saccharomyces cerevisiae sirtuin SIR2 has been extensively studied. It has many activities including silencing of mating-type loci, telomeric position effect silencing and silencing at the rDNA locus, suppression of illegitimate recombination and increasing cellular control of longevity. It is also implicated in response to dsDNA breaks.
Humans have seven sirtuins, although not all appear to have NDAC activity.
Human SIRT2 is a cytoplasmic, microtubule-associated protein. Is shows increases in abundance and phosphorylation at G2/M. It is a tubulin deacetylase. and is strongly down-regulated in many gliomas and glioma cell lines. Transgene replacement causes microtubule disruption and strongly reduces the number of stable clones expressing SIRT2 compared to a control in colony formation assays (Hiratsuka, M et al (2003) Biochem Biophys Res Commun. 309(3) 558-566).
Human SIRT3 is synthesized as an inactive proenzyme and activated by proteolysis on insertion into the mitochondrial matrix. Its function is unknown
Human SIRT1 is the closest human homologue to yeast SIR2. It is a nuclear protein found throughout the nucleus. Immunostaining of cells with anti-SIRT1 antibodies shows diffuse nuclear staining.
SIRT1 interacts with p53 via the p53 core and carboxy-terminus. It appears to act as a p53 deacetylase, as overexpression of SIRT1 results in reduced acetylation of p53. This in turn leads to reduced expression of endogenous p21, reduced transcription from a p21 reporter construct, and reduced apoptosis in response to H2O2 and γ-rays.
Overexpression of a catalytically inactive mutant SIRT1 enhances acetylation of p53, and sensitises cells to apoptosis induced by H2O2 and γ-rays.
The current consensus is that SIRT1 negatively regulates p53 function via deacetylation of p53, so that inhibition of SIRT1 function sensitises cells to p53-dependent apoptosis in response to cellular stress (Luo et al., 2001; Vaziri et al., 2001; Langley et al, 2002)
However, modulation of SIRT1 activity has until now been achieved by treatment of cells with the SIRT1 inhibitor nicotinamide, or by overexpression in trans of wild type and catalytically inactive forms of SIRT1. The use of nicotinamide is problematic, partly due to potential inhibition of the SIRT1-related NDACs and SIRT2 and SIRT3, and partly due to potential pleiotropic effects as nicotinamide is a natural cellular intermediary metabolite. Recent studies have also shown that transgene dosage may be critical in the analysis of p53 function (Blattner et al., 1999; Dumaz et al., 2001), and may underlie the sometimes-conflicting results that have been reported for p53 using this technique.